1. Evaluation of Enzyme Immunoassays to Detect Clostridium difficile Toxin from Anaerobic Stool CultureRosemary C. She, MD, Robert J. Durrant, MT, Cathy A. Petti, MDAm J ClinPathol 2009;131:81-84 JURNAL DIVISI PENYAKIT INFEKSI Ferdy/Aryati
2. Pendekatan diagnostik belum pasti C difficile Pendahuluan 2 Enzyme immunoassays (EIAs) untuk toksin A dan B mudah dilakukan, spesifik, hasil 24 jam Tes sensitif,spesifik dan cepatuntuk mendeteksi toksin masih dicari
3. Pendahuluan Isolat: pola kepekaan antibiotika, vaksin, danstrain typing Penting untuk diagnosis dan penatalaksanaan Kultur feses anaerob C difficile Tidak dapat membedakan toxin- producing dengan non–toxin-producing strains Tes sensitif, tapi tidak spesifik 3
4. Tes spesifik Pendahuluan 4 Cell culture cytotoxin neutralization bioassay (CTN) Butuh keterampilan Hasil lambat: 48 jam Konfirmasi strainyang menghasilkan toksin B dari spesimen langsung atau kultur
6. Pendahuluan Penelitian ini Membandingkan2 EIAkomersial dengan CTNuntuk mendeteksi toksinC difficiledari kultur. 6
7. SPESIMEN Spesimen feses dalam keadaan beku atau refrigerated (48 jam) Bahan dan Metode C difficile (ARUPLaboratories,Salt Lake City, UT) EIA vs CTN (91 isolat) Mei-Sept 2006 CTN (feses vs isolat) 115 sampel Nov 2007-Maret 2008 7
12. Bahan dan Metode 10 CTN Isolat +PBS Inokulasi pada 2 sumuran human foreskin fibroblast plate + MEM 2% (mengandung penicillin, streptomycin, and amphotericin B (Fungizone) Feses pengenceran 1:10
13. feses isolat Hasil Positif: CPE (+) pada sumuran tanpa antitoksin CPE (-) pada sumuran dengan antitoksin Bahan dan Metode DitambahC difficile goat antitoxin (TechLab) 11
19. Diskusi Standardisasi diagnosis C difficile–associated enteric diseasepenting pada institusi RS dan ahli epidemiologi Surveilans Pengendalian infeksi 17
24. Diskusi False negative EIA 10%: labilitas toksin C difficilekarena memerlukan kondisi lingkungan yang cocok Saran: isolat tidak disubkultur serial dan suspensi dipersiapkan secara khusus Penelitian ini:reprodusibilitas EIA dari isolat masih jelek Saran: algoritme tes EIA direvisi 22
25. Diskusi Fakta: tes in vitroproduksi toksin pada isolatC difficilelebih sensitif dibandingkan feses Berhubungan dengan banyaknya organisme yang menghasilkan toksin 23
26. Diskusi Walaupun reprodusibilitas EIA jelek namun positive predictive value tinggi mencerminkan jumlah toxin- producing > non–toxin-producing strains 24 Keterbatasan
27. Diskusi 2. Tidak dilakukan pemisahan toksin A dan toksin B. Peneliti tidak yakin keterbatasan ini mempengaruhi hasil penelitian karena tidak ada kasus hasil CTN negatif dan EIA positif. Penyakit manusia oleh C difficiledengan toksin A–positif/toksin B–negatifbelum diketahui 25
28. Diskusi 3. Tidak menggunakan metodenucleic-acid amplificationuntuk menganalisis ketidaksesuaian hasil EIA dan CTN. 26
29. Diskusi EIA dapat digunakan sebagai alat skrining cepat produksi toksin dari isolat Hasil negatif tidak dapat menyingkirkanadanya toksin EIA memiliki reprodusibilitas yang jelek dan EIA saja tidak dapat digunakan sebagai konfirmasi produksi toksin kultur anaerob, terutama pada klinis dengan prevalensi strain yang memproduksi toksin yang sedikit 27
32. CLOSTRIDIUM DIFFICILE AGARGeorge, W. L., V. L. Sutter, D. Citron, and S. M. Finegold. 1979. Selective and differential medium for isolation of Clostridium difficile. J. Clin. Microbiol. 9:214. Principles of the Procedure Proteose Peptone provides nitrogen, vitamins, and amino acids in Clostridium Difficile Agar. Fructose is the fermentable carbohydrate used to enhance recovery and growth of C. difficile. The Phosphates are buffering agents in this medium. Magnesium Sulfate is a source of inorganic ions to stimulate growth. Sodium Chloride maintains the osmotic balance of the medium. Agar is the solidifying agent. Horse blood provides essential growth factors(six amino acids for fermentative metabolism and growth—leucine, isoleucine, proline, tryptophan, valine, and glycine) in Clostridium Difficile Agar. Cycloserine and Cefoxitin are selective agents against aerobic, anaerobic, and facultatively anaerobic Gram-positive and Gram-negative bacteria. At the concentration of antibiotics used C. difficile is not inhibited significantly, while other anaerobes, including most clostridia, are inhibited.
33. Feses Feses for C difficile culture and toxin assays should be liquid, unformed; solid, formed stool or rectal swab are adequate to detect carrier’s but not to detect active cases of enterocolitis Stool should be placed in anaerobic transport containers and may be stored for up to 3 days at 4C or frozen -70C Text book microbiology-Baleyscot
34. BD™ Clostridium Difficile Agar with 7% Sheep Blood Freshly passed stool specimens (ideally 10 to 20 ml of watery stool) of patients suspected to suffer from pseudomembraneous colitis (PMC) should be used stool specimens must be fresh and should be processed within 2 hours if anaerobic transport media (charcoal transport swab/Anaerobic Transport Medium, CA) are not used
35. CLOSTRIDIUM DIFFICILE AGARGeorge, W. L., V. L. Sutter, D. Citron, and S. M. Finegold. 1979. Selective and differential medium for isolation of Clostridium difficile. J. Clin. Microbiol. 9:214. Cultural response on Clostridium Difficile Agar supplemented with 7% horse blood, cycloserine, and cefoxitin at 35°C after 24 - 72 hours incubation Colonies of C. difficileare 4 - 6 mm in diameter, irregular, raised, opaque, and grey-white after 48 hours incubation
36. Richard T. Ellison III, MDPublished inJournal Watch Infectious DiseasesMay 13, 2009 Which Toxin Causes C. difficile Disease? Clostridium difficile produces two virulence factors, toxins A and B, that have been implicated in the development of colitis. Although studies using purified toxins have suggested that C. difficile–associated disease (CDAD) is caused primarily by toxin A, clinical isolates from some patients with CDAD have yielded strains that produced only toxin B.
37. Clostridium difficile toxins A and B together are responsiblefor the symptoms of pseudomembranous colitis. Both toxinsintoxicate cultured cells by the same mechanism but theydiffer in cytotoxic potency, toxin B being generally 1,000 timesmore potent than toxin A. Don and T84 cells were used todetermine differences in the intoxication process exerted byboth toxins. Three main differences were identified: (a) the specific binding of radiolabeled toxins to the cell surfacescorrelated with the cytotoxic potency, (b ) toxin B was found to have a 100-fold higher enzymatic activity than toxin A, (c) toxin A was found to modify an additional substrate,Rap. Esteban Chaves-Olarte, et alMicrobiology and Tumorbiology Center (MTC), Stockholm, Sweden(J. Clin. Invest. 1997. 100:1734–1741.)
38. The authors’ concluded that a strain of C. difficile that was resistant tofluoroquinolones and had binary toxin and a partial deletionof the tcdC gene was responsible for this outbreak of CDADand that exposure to fluoroquinolones or cephalosporins was a risk factor. Review: Clostridium difficile-Associated Disorders/Diarrheaand Clostridium difficile Colitis: The Emergence of a MoreVirulent EraPerry Hookman · Jamie S. Barkin, Dig Dis Sci (2007) 52:1071–1075
39. Pathogenic C. difficile strains produce several known toxins. The most well-characterized areenterotoxin (toxin A) and cytotoxin (toxin B), both of which are responsible for the diarrhea and inflammation seen in infected patients, although their relative contributions have been debated.[2] Toxins A and B are glucosyltransferases that target and inactivate the Rho family of GTPases. Toxin A induces actin depolymerization by a mechanism correlated with a decrease in the ADP-ribosylation of the low molecular mass GTP-binding Rho proteins.[14] Another toxin, binary toxin, has also been described, but its role in disease is not yet fully understood.[15] Esteban Chaves-Olarte et al, Microbiology and Tumorbiology Center (MTC), Karolinska institutet, S-171 77 Stockholm, SwedenJ. Clin. Invest. The American Society for Clinical InvestigationVolume 100, Number 7, October 1997, 1734–1741
40. Pathogenic strains of C. difficile causes disease by the release of two protein enterotoxins, toxin A and toxin B, which cause severe inflammation and mucosal injury to the colon--colitis. At this point, the toxins attract white blood cells to the area, and the white blood cells may provide a protective immune response. However, if the white blood cells do not provide the desired immune response, mild CDAD occurs. Nevertheless, the toxins can actually kill the lining of the intestine, causing it to fall off and mix with the white blood cells and give the appearance of yellow plaques (patches) in the intestines. This is called pseudomembranous colitis because the patches look like membranes but aren’t true membranes. Johnson, M. T. 2009. Medical Microbiology <URL: http://web.indstate.edu/thcme/micro/anaercult/sld022.htm>. Accessed 7 April 2009.
41. Two toxins, A and B, are involved in the pathogenesisof CDAD [20–25]. These toxins A and B act by disruptingthe actin-cytoskeleton of fibroblasts in tissue culture [25]and in intestinal epithelial cells by uridine 5-diphosphateglucose-dependent glucosylation of Rho proteins [26]. Thisis associated with the toxin entering the cell. An inflammatorycascade is then initiated vianeuroimmune mediators,specifically substance P and mast cell products with release of histamine leukotrienes. Kim et al. [27] recently documented that toxin A alsocauses marked apoptosis of colonocytes and contributes tothe formation of colonic ulcers and pseudomembranes. Itdoes so through a signaling pathway that involves p38-dependent activation of p53 and subsequent induction ofp21, which then results in cytochrome c release and caspase-3 activation through Bak induction Review: Clostridium difficile-Associated Disorders/Diarrheaand Clostridium difficile Colitis: The Emergence of a MoreVirulent EraPerry Hookman · Jamie S. Barkin, Dig Dis Sci (2007) 52:1071–1075
42. Am J PhysiolGastrointest Liver Physiol 285: G1049–G1055, 2003. Toxin A induces the synthesis of proinflammatorycytokines such as IL-1, TNF-, or IL-8 in macrophages,lymphocytes, or intestinal epithelial cells (5,11). In animal models, toxin A is responsible for neurallymediated gut inflammation and diarrhea. It hasbeen determined that inflammation induced by toxin Ais mediated via tachykinergic pathways (6, 20, 21).Diarrhea induced by toxin A was significantly reducedby the blockade of neuronal nicotinergic pathways Toxin B of C. difficile is more potent than toxin A (11,28), but few data are available concerning its effects onthe human gut. Toxin B can increase intestinal epithelialbarrier permeability (28) and induce the synthesisof proinflammatory cytokines in monocytes(11). Nevertheless,it is presently unknown whether toxin B cancause the synthesis of proinflammatory cytokines oractivate enteric neuronal pathways in the human colon
43. Pathogenesis of Clostridium difficile-Associated Disease ED/LS. 2007. Southend University Hospital <URL:http://www.southend.nhs.uk/Hospital+Services/Other+Services/Infection+Control/Clostridium+Difficile/>. Accessed 7 April 2007
44. Clostridium difficile is a species of Gram-positive, rod-shaped, spore-forming bacteria. C. difficile are anaerobic—lives in the absence of oxygen. In the presence of oxygen, the vegetative form of C. difficile can survive up to 24 hours on an inanimate surface; whereas, C. difficile spores can survive up to 2 years on inanimate surfaces that are exposed to oxygen. C. difficile moves via peritrichous flagella. These flagella are evenly spread around the surface of each cell and allow the bacteria to be highly motile. The bacteria are able to move by a tumbling motion and by movement in a forward direction. Savita Shrivastava. 2007 BacMap <URL: http://wishart.biology.ualberta.ca/BacMap/graphs_cgview.html>. Medical Microbiology 54: 97-100, Accessed 7 April 2009
45. C. difficile is heterotrophic which means that it cannot make its own food and relies on organic substances for nutrition. Since C. difficile is anaerobic, it obtains its energy, ATP, by fermentation of carbon and nitrogen substrates. C. difficile requires six amino acids for fermentative metabolism and growth—leucine, isoleucine, proline, tryptophan, valine, and glycine. Poxton, I. R. 2005. Clostridium difficile. Journal of Medical Microbiology 54: 97-100
46. Biochemical reactions C difficile: Lechitinase C activity: negative Lipolysis: negative Lactose fermentation: negative Proteolysis: negative
47. Merkel. 2006. Microbe World. <URL:http://www.microbeworld.org/know/spore.aspx>. Accessed 7 April 2009.
48. Flow chart for two-step testing algorithm to detect toxigenicC. difficile.JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 2006, p. 1145–1149 46
49. European Society of Clinical Microbiology and Infectious Diseases(ESCMID): Data review and recommendations for diagnosingClostridium difficile-infection (CDI), Clin Microbiol Infect 2009; 15: 1053–1066
50. Binomial Test In statistics, the binomial test is an exact test of the statistical significance of deviations from a theoretically expected distribution of observations into two categories. The most common use of the binomial test is in the case where the null hypothesis is that two categories are equally likely to occur. Tables are widely available to give the significance observed numbers of observations in the categories for this case
51. JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 2005, p. 5338–5340 Clostridium difficile-associated diarrhea (CDAD) is the mostimportant infectious cause of nosocomial diarrhea andpseudomembranous colitis. The enteropathogenicity dependson the production of enterotoxin A (308 kDa) and cytotoxin B(270 kDa). Several authors have suggested that all fecal samplesfor C. difficile from patients with diarrhea hospitalized formore than 72 h be investigated (3) irrespective of the physician’srequest 49
52. infection control and hospital epidemiology february 2007, vol. 28, no. 2 A CDAD case is defined as a case of diarrhea (ie, unformed stool that conforms to the shape of a specimen collection container) or toxic megacolon (ie, abnormal dilation of the large intestine documented radiologically) without other known etiology that meets 1 or more of the following criteria: (1) the stool sample yields a positive result for a laboratory assay for C. difficile toxin A and/or B, or a toxin-producing. C. difficile organism is detected in the stool sample by culture or other means; (2) pseudomembranous colitis is seen during endoscopic examination or surgery; and (3) pseudomembranous colitis is seen during histopathological examination. The CDAD case definition may be implemented for laboratory-based reporting systems by focusing only on criterion 1, if the laboratory routinely performs tests for C. difficile only on unformed stools. 50
53. infection control and hospital epidemiology february 2007, vol. 28, no. 2 A recurrent CDAD case is defined as an episode of CDAD (ie, one that meets the criteria for a CDAD case) that occurs 8 weeks or less after the onset of a previous episode, provided that CDAD symptoms from the earlier episode resolved with or without therapy. The recurrent CDAD case definition may be implemented for laboratory-based reporting systems on the basis of the following stipulations: (1) an additional positive result of a laboratory test performed on a specimen collected 2 weeks or less after the last specimen that tested positive represents continuation of the same CDAD case, (2) an additional positive result of a laboratory test performed on a specimen collected 2-8 weeks after the last specimen that tested positive represents a recurrent CDAD case, and (3) an additional positive result of a laboratory test performed on a specimen collected more than 8 weeks after the last specimen that tested positive represents a new CDAD case. 51
54. infection control and hospital epidemiology february 2007, vol. 28, no. 2 A case patient with severe CDAD is defined as a case patient who meets any of the following criteria within 30 days after CDAD symptom onset (or, in the case of laboratory-based reporting, within 30 days after the index laboratory test): (1) history of admission to an intensive care unit for complications associated with CDAD (eg, for shock that requires vasopressor therapy); (2) history of surgery (eg, colectomy) for toxic megacolon, perforation, or refractory colitis; and (3) death caused by CDAD within 30 days after symptom onset (eg, as listed on the death certificate or recorded in the medical record by a clinician caring for the patient) 52
55. Ann Intern Med. 2006;145:758-764. The most common inducing agents have been clindamycin or broad-spectrumcephalosporins, but nearly all agents with an antibacterial spectrum may be responsible Standard treatment of C. difficile infection includes withdrawal ofthe inducing agent and use of oral metronidazole or oralvancomycin; metronidazole is preferred in guidelines, butvancomycin is probably more effective, especially in seriously ill patients. 53
57. JOURNAL OF CLINICAL MICROBIOLOGY,Jan. 1998, p. 184–190 55 Cell-rounding reaction caused in Chinese hamster ovary K-1 cells byC. difficile toxins A and B. (A) Cell-rounding activity typical of both toxins. ToxinB masks the activity of toxin A because of its high specific activity in the assay. (B) Neutralization of cell-rounding activity with specific C. difficile antitoxin. The activity is not neutralized with nonimmune serum. (C) Cell-stretching that may be confused with the cytotoxic activity of C. difficile toxins A and B.
58. JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 1998, p. 2178–2182 Comparison of cytotoxic effects of a toxin A-, toxin B+ strain withthose of a toxin A+, toxin B+ strain on a Vero cell sheet. Filtrates of C. difficile strains cultured in brain heart infusion were applied to monolayers of Vero cells. Normal Vero cell monolayer; Vero cells treated with culture filtrate from toxin A+, toxin B+ strain; Vero cells treated with culture filtrate from toxin A-, toxin B+ strain.
59. JOURNAL OF CLINICAL MICROBIOLOGY,Jan. 1998, p. 184–190 Culture. Prereducedanaerobically sterilized brain heart infusion broth (Carr-Scarborough, Stone Mountain, Ga.) was supplemented with cycloserine andcefoxitin (CC-BHI) (Oxoid, Ogdensburg, N.Y.). The final concentrations ofcycloserine and cefoxitin were 500 mg/ml and 16 mg/ml, respectively. For inoculationof broth media, 1 drop of stool specimen was added to a tube containing5.0 ml of supplemented broth. Inoculated media were incubated at 37°C for 3 to4 days. Selective cycloserine-cefoxitin-fructose agar (11) (CCFA) (Oxoid) wasprepared as recommended by the manufacturer. Presumptive colonies werecharacterized by a yellowish color, flat morphology, yellow-green fluorescence,and a horsey smell. CCFA plates were incubated anaerobically at 37°C for aminimum of 3 days. Selected strains were grown in brain heart infusion dialysis flasks as previously described 57
60. A New Rapid Test for Detecting Clostridium difficile Toxins A and B in Fecal SpecimensD. M. Lyerly1, J. T. Boone1, L. Zheng1, C. W. Genheimer1, S. Keller1, K. Long2, D. Taniguchi21TECHLABÒ, Inc., Blacksburg, VA and 2West Virginia University Hospitals, Morgantown, WV 58
61. A New Rapid Test for Detecting Clostridium difficile Toxins A and B in Fecal SpecimensD. M. Lyerly1, J. T. Boone1, L. Zheng1, C. W. Genheimer1, S. Keller1, K. Long2, D. Taniguchi21TECHLABÒ, Inc., Blacksburg, VA and 2West Virginia University Hospitals, Morgantown, WV
62. A variety of normal intestinal bacteria and intestinal pathogens, including bacteria, viruses, andparasites, were checked for cross-reactivity in the TOX A/B QUIK CHEKTM. Only toxigenic C. sordellii,which produces toxins HT (hemorrhagic toxin) and LT (lethal toxin) that are immunologically related totoxins A and B, reacted in the test A New Rapid Test for Detecting Clostridium difficile Toxins A and B in Fecal SpecimensD. M. Lyerly1, J. T. Boone1, L. Zheng1, C. W. Genheimer1, S. Keller1, K. Long2, D. Taniguchi21TECHLABÒ, Inc., Blacksburg, VA and 2West Virginia University Hospitals, Morgantown, WV
63.
64. EIA Principle of Procedure During the first incubation, C. difficile toxins A+B present in the stool supernatant are captured byantibodies attached to the wells. The second incubation adds additional anti-toxin A+B antibodies that"sandwiches" the antigen. The next incubation adds an anti-second antibody conjugated to peroxidase. After washings to remove unbound enzyme, a chromogen is added which develops a blue color in thepresence of the enzyme complex and peroxide. The stop solution ends the reaction and turns the blue color to yellow. Zero reader on air. Read all wells at 450/620-650 nm.
67. EIAJOURNAL OF CLINICAL MICROBIOLOGY, Dec. 1991, p. 2724-2730 Test performance. Following equilibration at roomtemperature, the required number of microwells (one wellfor each specimen plus one positive and one negative controlwell for each batch) were removed from the protectivepouch. Microwells were placed in a 96-microwell stripholder, and one drop of enzyme conjugate (50 pi) was addedto all wells. The diluted stool samples (50 pul) were thenadded to appropriate wells. Following the addition of positiveand negative controls to the designated wells, the platewas mixed by gently shaking it for 30 s. Plates were sealedand incubated for 2 h at 35°C.
68. JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 1991, p. 2724-2730 All microwells were washedfive times with a squirt bottle containing wash buffer. Afterremoval of the final wash buffer, one free-falling drop ofsubstrate A was added to each well; this was followed by theaddition of a drop of substrate B. The plate was gentlyshaken for 15 s to mix the substrates and was then incubatedfor 10 min at room temperature. One drop of stop solutionwas added to all wells, and the plate was gently shaken for 15s to mix the substrates. The initial color of a positive reactionwas blue; this changed to yellow upon the addition of the stop solution. Reading Plates were read visually within 15 min of theaddition of the stop solution. Plates were read within 30 minby using an EIA reader (450 nm) zeroed on air. A reading of<0.100 was negative, a reading of .0.100 but <0.150 wasindeterminate, and a reading of .0.150 was positive.
69. JOURNAL OF CLINICAL MICROBIOLOGY,0095-1137/98/$04.0010Apr. 1998, p. 1167–1168 In contrast, very few cases of C. difficilebacteremia or other extraintestinalillness have been reported (2, 6, 7), and these have occurredmainly when a severe underlying disease was present. In mostcases of septicemia, the source of infection has been found tobe the gastrointestinal tract, as suggested by isolation of C.difficile with other intestinal bacteria (9).
70. Serum Ig G,A,M C dif(N Engl J Med 2000;342:390-7.) Levels of antibody against C. Difficile toxin A, toxin B, and the nontoxinantigen preparation were measured by an enzyme-linkedimmunosorbentassay (ELISA) as previously described.13,17,19 Serum samples or filter-sterilized fecal samples from a different populationof patients, with high levels of antitoxinimmunoglobulin(IgG, IgA, or IgM) were pooled, assigned an arbitrary value thatwas expressed in ELISA units, and used in all assays as s
73. Proposed algorithm for detection of C. difficile in fecal specimens submitted for C. difficile testingJOURNAL OF CLINICAL MICROBIOLOGY, Jan. 2008, p. 328–330 71
75. Voth DE, et al. Clinical Microbiology Reviews 2005; 18(2) 247-263Genetic Loss of Regulation: NAP1/BI/027
76. Voth DE, et al. Clinical Microbiology Reviews 2005; 18(2) 247-263Genetic Loss of Regulation: NAP1/BI/027
77.
78. Evaluation of a test for Clostridium difficile toxins A and B for the diagnosisof neonatal swine enteritisKaren W. Post, B. Helen Jost, J. Glenn SongerJ Vet Diagn Invest 14:258–259 (2002) The EIA was conducted according to the manufacturer’sdirections using kits from a single lot. The test protocol includesmicrowells with antibodies to C. difficile toxins A andB. Test wells are coated with polyclonal goat antibodyagainst the toxins. The detecting antibody is a mixture oftoxin A monoclonal mouse antibody and toxin B polyclonalgoat antibody, both conjugated to horseradish peroxidase. An aliquot of either colonic contents or feces was emulsifiedin the kit diluent. One hundred microliters of diluted specimenwas then transferred to a microtiter well. Any toxinspresent in the specimen bound to the well, which subsequentlybound the detecting antibodies. Upon the additionof substrate, a color change occurred in the wells with enzyme–antibody–antigen complexes formed in the presenceof toxin. Results were read visually against a white background,and any yellow color was interpreted as a positivereaction. Positive and negative controls were performed witheach series of test specimens.
